Breeding of Solanum lycopersicum aims at the production of commercial varieties optimally adapted to growing and storage conditions. A challenge breeders are facing is finding an improved balance between fruit firmness post-harvest and consumer desires in terms of taste, texture and colour. These consumer desires relate strongly to fruit ripening. Fruit ripening is a complex developmental process responsible for the transformation of the seed-containing organ into a tissue attractive to seed dispersers and agricultural consumers. The changes associated with fruit ripening, in particular post-harvest softening, limit the shelf life of fresh tomatoes.
For tomato fruit growth and development, a number of consecutive phases can be discerned: floral development, pollination, then early fruit development takes place which is characterised by a high frequency of cell division and the fruit is rapidly increasing in size mainly due to cell expansion. At the end of the third phase the fruit reaches the mature green stage. During the fourth phase, fruit ripening takes place which is characterised by a change in colour and flavour as well as fruit firmness and texture.
The build-up of the characteristic red colour of the tomato fruit is caused by the accumulation of lycopene and carotene. In general, different colouration phases are distinguished: mature green, breaker, pink and red. At the breaker stage, the typical red pigmentation initiates. Red ripe stage or red ripe harvested fruit stage is the stage where the fruit has reached its mature colour on the major part of the fruit.
In addition to the colour changes, during fruit ripening enzymatic activity leads to degradation of the middle lamellar region of the cell walls which leads to cell loosening which is manifested as softening and loss of texture of the fruit. Softening of the fruit is often measured as external resistance to compression which can be quantified for example by a penetrometer.
Modification of single genes known to be involved in ripening has not yet resulted in a fruit with normal ripening but minimal tissue softening.
Ripening and senescence in climacteric fruits such as tomatoes are promoted by ethylene. Ethylene is autocatalytic for its own biosynthesis through increases in 1-Aminocyclopropae-1-carboxilic acid (ACC) synthase (ACS) and ACC oxidase (ACO). ACS is also referred to as 1-aminocyclopropane-1-carboxylate synthase; Le-ACS; or S-adenosyl-L-methionine methylthioadenosine-lyase. An increase in the amount of ACS and ACO thus leads to an increased conversion of L-methionine into ethylene. At least eight ACS genes (LEACS1A, LEACS1B, and LEACS2-7) have been identified in tomato (Alexander et. al., Journal of Experimental Botany, Vol 53, No 377, pp 2039-2055, 2002) and each ACS has a different expression pattern.
ACC synthase (ACS) is an enzyme that catalyzes the synthesis of 1-aminocyclopropane-1-carboxylic acid (ACC) from S-Adenosyl methionine. ACC is then converted into ethylene catalyzed by ACO. The biosynthesis of ethylene is for example described by Stearns and Glick (Biotechnology Advances 2003, vol 21 pp 193-210), which is enclosed by reference.
ACS belongs to the α-family of pyridoxal-5′-phosphate (PLP) dependent enzymes and shares a modest level of similarity with other members of this family like aspartate amino-transferase (AATase and tyrosine aminotransferase (TATase). The structure of ACS from various sources has been described by Capitani et al. In a sequence alignment of eight ACS proteins (Malus domestica, Phaseolus aureus, Solanum tuberosum, Pelargonium hortorum, Nicotiana tabacum, Cucumis melo, Lycopersicon esculentum, and Brassica oleracea) they describe conserved regions which are indicated in FIG. 1 in this Capitani publication. (Capitani et al., Journal of Molecular Biology, 1999, vol 294, pp 745-756).
Two systems have been proposed to operate in climacteric plants regulating ethylene production. The first is functional during normal vegetative growth (system 1); it is auto inhibitory and responsible for production of basal ethylene levels that are detected in all tissues including those in non-climacteric plants. System 1 continues during fruit development until a competence to fruit ripening is attained. Then a transition period is reached wherein LEACS1A and LEACS4 are activated resulting in an increased level of ethylene. This increased ethylene level induces the expression of LEACS2 which starts system 2 which is active during the ripening of climacteric fruit. In system 2, ethylene production is auto catalytic. This complexity of the ethylene regulation has been studied using antisense inhibition of LEACS2 in transgenic plants (Barry et al., Plant Physiology vol 123, pp 979-986, 2000).
WO2005/016504 discloses “stay green” plants, i.e. a plant phenotype whereby leaf senescence is delayed compared to a standard reference. It discloses plants with disrupted ACS2, ACS6, ASC7 genes which disruption inhibits the expression or activity of said ACS.
Yokotani et al describe transgenic tomatoes with all known LeEIL genes (Ethylene Insensitive Like genes) suppressed to study the regulatory mechanisms of ethylene biosynthesis (Yokotani et al, Journal of Experimental Botany, vol 60, pp 3433-3442, 2009).
ACS2 mutants are known for instance from Vijee Mohan et al. They disclose (Vijee Mohan et al. Sol 2010, page 67 world wide web at so12010.org/so12010/files/file/SOL2010_Abstracts.pdf) EcoTILLING in tomato wherein nucleotide sequence diversity is used as a measure of the genetic variation that is present in a species, especially Single Nucleotide Polymorphisms (SNPs) are used to represent the most common variations across a genome. EcoTILLING is used as a high throughput, low cost technique for rapid discovery of polymorphisms in natural populations by heteroduplex analysis using a mismatch-specific endonuclease. A collection of tomato accessions was obtained from different sources like NBPGR (India), IIVR (India) and TGRC (California, USA) and were analyzed for the frequency of naturally occurring SNPs, among others in ACS2. A number of SNPs were detected for different genes but with a varying frequency. The morphological features during different developmental stages and chemotypic observations indicated wide variations among the accessions. Correlations between these variations with the location of the SNPs is suggested to be useful in finding promising alleles for crop development.
Studies on transgenic tomato fruit (ACS2 mutants) in which ethylene production was suppressed (Oeller et al, 1991 Science, vol 254, pp 437-439); (Picton et al. 1993 The Plant Journal vol 3 pp 469-481) showed delayed fruit ripening and revealed a critical role of ethylene in fruit ripening.
WO 92/04456 discloses control of plant development characteristics effected by ethylene in tomato and zuchini, among others by recombinant production of ACS and by using antisense technology or mutated ACS genes.
Despite the above, no useful alleles for tomato fruit ripening improvement have been found so far.
There is thus a need for cultivated tomato plants with a modified ethylene production having a delayed ripening and/or longer shelf-life of the tomato fruits compared to wild type tomato plants.